Human immunoglobulin G (IgG) antibodies exist in four subclasses with distinct structural and functional properties. IgGs are composed of two heavy chain-light chains pairs (half-molecules), which are connected via inter-heavy chain disulfide bonds situated in the hinge region. Human IgG4 molecules exist in various molecular forms which differ by the absence or presence of the inter-heavy chain disulfide bonds located in the hinge region. IgG4 molecules exist in forms in which either both or none of the inter-heavy chain disulfide bonds have been formed (6, 7). However, irrespective of the absence or presence of these inter-chain disulfide bonds (6, 8), human IgG4s exist as tetramers in solution consisting of two Ig heavy and two light chains, as common for immunoglobulin G molecules, due to relatively strong non-covalent interactions between the CH3-domains and between the CH1 and CH2 domains (4). Only upon denaturation under non-reducing conditions, the two non-covalently associated half molecules dissociate as demonstrated by size-determination analysis such as SDS-PAGE (6, 9).
It has been known for several years that human IgG4 antibodies, unlike other IgG subclasses, behave as monovalent molecules in interactions with antigen. It was found that serum-derived human IgG4 cannot precipitate purified antigen, because it cannot crosslink. While such serum-derived IgG4 is functionally monovalent (1, 2), recombinantly produced IgG4, in contrast, is behaving bivalently in interactions with antigens (3). On the basis of these observations, it has been proposed that IgG4 molecules in serum can exchange half-molecules (i.e. a molecule consisting of one heavy chain and one light chain), resulting in the generation of bispecific molecules, which cannot crosslink identical antigens (3-5). This process of half-molecule exchange is also termed “Fab-arm exchange” herein.
Bispecific antibodies have interesting potential as therapeutic drugs, since they can be used, for example, as mediators to retarget effector mechanisms to disease-associated sites. However, one of the major obstacles in the development of bispecific antibodies has been the difficulty of producing the materials in sufficient quality and quantity by traditional technologies, such as the hybrid hybridoma and chemical conjugation methods (10).
WO 2005/062916 describes methods for the formation of multimeric molecules on the basis of IgG4 in vivo in mice. Furthermore, WO 2005/062916 describes that co-incubation of two IgG4 antibodies having different antigen-binding specificities in vitro in a saline buffer leads to the formation of products that are capable of reacting with both antigens. However, it has not been demonstrated in WO 2005/062916 whether these products are aggregates or bispecific antibodies, and the yield of the reaction was low under the conditions used.